RU2492241C1 - METHOD OF PRODUCTION OF PROTECTIVE ANTIGEN AND PROTEIN OF S-LAYER EA1OF ASPOROGENOUS RECOMBINANT STRAIN IN anthracis 55 ΔTPA-1Spo- - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method includes cultivation of previously prepared culture of the recombinant strain B. anthracis 55ΔTPA-1Spo^-. The cell mass is separated using the filtration module with a membrane having a pore diameter of 0.2 mcm. Protein EA1 is extracted from the washed cell mass using a buffer with 1% sodium dodecyl sulfate, and purified by diafiltration using membrane filters and two-stage ion-exchange chromatography on hydroxyapatite. The protective antigen is isolated from the culture filtrate and purified by successive steps of concentration and diafiltration.

EFFECT: use of the invention enables to obtain in one processing chain the highly purified antigens of anthrax microbe - protective antigen and protein EA1 needed to create chemical vaccines.

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Description

The invention relates to biotechnology, and in particular to a technology for producing immunogenic anthrax antigens - a protective antigen and EA1 protein, and can be used in the production of specific prophylaxis of anthrax.

B. anthracis is the etiological agent of a particularly dangerous infectious disease - anthrax. The high virulence of the anthrax pathogen is due to genes that determine the synthesis of capsules and exotoxin. The capsule protects the microbial cell from phagocytosis, and the exotoxin, consisting of a protective antigen, edematous and lethal factors, causes pathological reactions of the macroorganism. One of the components of the toxin is a protective antigen, has a pronounced immunogenic potential and is the active principle of all anthrax vaccines [1]. Despite the dominant importance of the protective antigen in the process of formation of the immune response, participation in the immunogenesis of other antigens, including the S-layer protein - EA1 [2], is not questioned. The introduction of additional immunogenic antigens into the vaccine is one of the approaches to increasing their immunological effectiveness.

A combined vaccine is being produced in Russia, which is a composition of the spores of the vaccine strain B. anthracis STI-1 and the protective antigen isolated from it adsorbed on aluminum hydroxide. A chemical vaccine is licensed in the UK consisting of an alum-precipitated protective antigen isolated from a culture filtrate of the vaccine strain B. anthracis Sterne 34F ₂ . A US licensed chemical vaccine contains a protective antigen adsorbed onto aluminum hydroxide from a culture filtrate of a proteasonegative derivative of B. anthracis Sterne 34F ₂ - B. anthracis V770-NP1-R.

Known methods for producing protective antigen from vaccine strains of B. anthracis STI-1, B. anthracis Sterne 34F ₂ , B. anthracis V770-NP1-R [3, 4, 5]. A known method of obtaining a protective antigen from strain B. anthracis 55 VNIIIVViM used in veterinary medicine [6]. In all of the above methods, producers of the anthrax protective antigen are strains that are similar to each other by the production method - elimination of plasmid pXO2, and plasmid composition - pXO1 ⁺ pXO2 ^- . Due to the absence of the pXO2 plasmid, they lack the ability to synthesize the capsule and, as a result, cause an infectious process when it enters a susceptible organism. However, these strains are characterized by relatively high reactogenicity, caused, among other things, by the pathogenicity factors they synthesize - edematous and lethal factors. In addition, they, like all typical representatives of the genus Bacillus, form spores that are resistant to most disinfectants. The use of spore-forming microorganisms in the industrial production of vaccine preparations carries a risk of bacterial contamination of equipment and facilities.

Another disadvantage of methods using vaccine producer strains is their inability to provide a high level of protective antigen production due to genetic characteristics, namely the presence of a complex regulatory system in the B. anthracis genome that limits its synthesis [7, 8]. The level of production of protective antigen by attenuated strains depends on the content of CO ₂ in the atmosphere and is no more than 15-30 μg / ml. To increase the production of protective antigen, a method has been developed consisting in the deep cultivation of the B. anthracis STI-1 strain on a nutrient medium with sodium bicarbonate with continuous aeration of the culture fluid with a mixture of several gases - nitrogen, argon, helium [9]. However, the use of a mixture of different gases for aeration unnecessarily complicates the technology of cultivation. The increased content of CO ₂ in the growing medium is achieved by adding sodium bicarbonate to it. However, it was found that under these conditions, the proportion of cells with mutations in the pag gene encoding its synthesis increases in the microbial population. There is a method that offers the addition of sodium bicarbonate to the medium at a certain stage of the growth of the microorganism [10]. This leads to a more stable production of protective antigen, but does not change the possible limit of its synthesis, due to the characteristics of the genotype.

All of the above methods for producing a protective antigen from vaccine strains involve the preparation of a uterine inoculum culture of strain B. anthracis; deep anaerobic cultivation in a nutrient medium containing sodium bicarbonate; concentrated and sterilization of the culture filtrate; precipitation of the protective antigen present in the filtrate on an aluminum hydroxide gel or similar adjuvant. However, they do not include the stages of thin chromatographic purification, as a result of which the resulting product has an indefinite composition and contains a large amount of ballast substances, which does not correspond to the modern requirements presented to preventive drugs. In addition, the protective antigen obtained by such methods, even when the chromatography step is turned on, will contain impurities of edematous and lethal factors, due to the complexity of the chromatographic separation of proteins with very close molecular weight values. The presence of unnormalized amounts of reactogenic components of anthrax toxin in chemical vaccines can lead to the development of post-vaccination complications.

Today, methods for producing a protective antigen from genetically engineered producers with the cloned pag gene determining its synthesis have an advantage.

Known methods for producing protective antigen from recombinant strains based on bacillary strains of B. anthracis and B. subtilis [11, 12]. These methods use genetically engineered producers containing hybrid plasmids with the pag gene included in their composition. These strains do not synthesize the pathogenicity factors of the anthrax microbe - the capsule, edematous and lethal factors, and are therefore safe. In addition, they have low activity of proteolytic enzymes, as a result, the levels of production of protective antigen reach high values - 100-120 μg / ml. Methods for producing a protective antigen from recombinant bacillary strains include preparing a seed culture; cultivation under normal conditions in a nutrient medium with intensive mixing for aeration; concentrated and sterilization of the culture filtrate; isolation and chromatographic purification of protective antigen. However, the disadvantage of the proposed technologies is the formation in the process of cultivation of microbial spores that are resistant to ultraviolet radiation, disinfectants and stored in the environment for many decades.

Known methods for producing anthrax protective antigen from Escherichia coli strains containing the cloned pag gene [13, 14]. However, unlike representatives of the genus Bacillus, which are capable of exogenous protein synthesis, E. coli is characterized by an intracellular mechanism of secretion of protective antigen, which negatively affects the amount of the target product obtained, and, in addition, complicates its isolation and purification. For the production of a non-toxic anti-anthrax vaccine, a procedure was proposed for obtaining a protective antigen from E. coli strain DH5α with the cloned pag gene [15]. The increase in the yield of protein antigen was achieved by improving the cultivation process in the fermenter; polyols, carbohydrates, organic acids and mineral salts were used as additives to the standard medium. However, a change in technology due to the introduction of significant amendments to the composition of the cultivation medium reduces its profitability.

A known method of creating a vaccine from anthrax protective antigen [16]. This patent describes a method for producing a protective antigen from an asporogenous recombinant bacillary strain. The method includes preparing a seed culture of the producer; growing at a constant temperature of 37 ± 0.1 ° C until a stationary growth phase is reached; filtration through a bacterial filter (0.2 μm); concentrated culture filtrate and diafiltration; isolation and purification of protective antigen on a chromatographic system of high pressure using ion-exchange carriers. However, in the proposed method, the B. anthracis DELTA Sterne-1 (pPA102) CR4 strain, obtained on the basis of the plasmid-free derivative of the vaccine strain B. anthracis Sterne, which has high proteolytic enzyme activity [17], which is only partially stopped by ingredients of the growth medium [18]. The proteolytic effect of the producing microorganism is a serious problem for the organization of technological lines for the production of biologically significant antigens. In addition, a scalable cultivation of the recombinant strain of B. anthracis DELTA Sterne-1 (pPA102) CR4 is carried out on a nutrient medium with the addition of a selective antibiotic in a relatively high concentration of 40 μg / ml. The use of an antibiotic in the technology for producing a bacterial antigen, which is part of the drug for vaccinating people, is undesirable. In addition to all of the above, the producer underlying the method is not available to Russian manufacturers.

A known method for producing protein of the S-layer of anthrax microbe EA1 [19]. The method includes the following steps: growing strain B. anthracis V770-NP-1R (pXO1 ⁺ pXO2 ^- ) in a liquid nutrient medium (R medium) for 18-20 hours, sedimentation of the cell mass by centrifugation, disintegration of cell walls, incubation with guanidine hydrochloride for 2 hours at room temperature, precipitation of the guanidine extract with alcohol, followed by washing with distilled water. However, the final product obtained using this method contains, in addition to EA1, numerous associated proteins, which does not meet the requirements for a modern chemical vaccine.

The analysis of the prior art, including a search by patents and scientific and technical sources of information, showed that no information was found on a method that combines the preparation of a protective antigen and EA1 protein in one technological process.

The objective of the invention is to develop a biologically safe and effective method for producing preparative quantities of highly purified antigens of anthrax microbe - protective antigen and EA1 protein, necessary for creating chemical vaccines.

The technical result consists in the possibility of obtaining in one technological chain preparative amounts of highly immunogenic preparations of a protective antigen and EA1 protein of anthrax microbe, which effectively protect against anthrax causative agent, interacting with innate immunity structures and are not toxic to a macroorganism.

The technical result is achieved by a method of obtaining a protective antigen and protein EA1 of the anthrax microbe, including the preparation of an inoculum culture of the producer strain; cultivation of an asporogenic recombinant strain of B. anthracis 55ΔTPA-1Spo ^- at a temperature of 37 ± 0.1 ° C for 16 hours in a liquid nutrient medium containing tryptone with a concentration of at least 20 mg / ml, with constant stirring at 150 rpm; separating the cell mass using a filtration module with a membrane having a pore diameter of 0.2 μm; Isolation of EA1 protein from the washed cell mass and its purification using successive incubation steps with extraction buffer containing 1% sodium dodecyl sulfate, diafiltration using membrane filters with an exclusion limit of proteins with a molecular weight of 30 kDa and two-stage ion-exchange chromatography on hydroxyapatite; isolation of protective antigen from the culture filtrate and its purification using successive stages of concentration and diafiltration using membrane filters with an exclusion limit of proteins with a molecular weight of 30 kDa and two-stage chromatography on an ion-exchange carrier and a gel filtration carrier.

In the claimed method, as an producer of the protective antigen and protein of the S-layer (EA1) of the anthrax microbe, an asporogenous and avirulent genetically engineered strain of B. anthracis 55ΔTPA-1Spo ^{- is used} . The strain was developed by the Federal State-Funded Educational Institution of the Russian Research Institute "Microbe" and is protected by RF patent No. 2321629. The strain was created by genetic engineering on the basis of the plasmid-free derivative of the vaccine strain B. anthracis 55, it retains biological properties when cultured on nutrient media and stored, it does not contain determinants of the synthesis of the main factors of virulence of the causative agent of anthrax, and the presence of chromosomal mutations determines the asporogenic phenotype. Using asporogenous avirulent B. anthracis 55ΔTPA-1Spo ^- to produce protective antigen and protein EA1 anthrax microbe provides biological safety works and eliminates the risk of contamination of premises and equipment anthrax pathogen.

The high level of production of the protective antigen of the anthrax microbe is ensured by the functioning of the cloned pag gene in the multicopy hybrid plasmid. Strain B. anthracis 55ΔTPA-1Spo ^- synthesizes 10-20 times more protective antigen than the vaccine strain of B. anthracis STI-1, due to its lack of a negative regulator of the synthesis of protective antigen pagR and increase the number of gene copies encoding pag due multicopy vector plasmid . For the effectiveness of the procedure for isolation and purification of the protective antigen and EA1 protein, it is extremely important that the strain B. anthracis 55ΔTPA-1Spo ^{- is} characterized by a sharply reduced activity of proteolytic enzymes. The proposed producer of anthrax antigens grows well both in traditional domestic media, for example, Hottinger broth, and in standardized semisynthetic media with a high content of tryptone. Adding tryptone to Hottinger broth at a concentration of up to 20 mg / ml increases the production of protective antigen by 1.5-2 times. The experimentally established by the applicant growing time of 16 hours leads to an increase of about 5 times the yield of protective antigen. An essential point for creating vaccines is that the cultivation of the genetically engineered strain of B. anthracis 55ΔTPA-1Spo ^- for 16 hours can be carried out without the addition of a selective antibiotic. It was found that during this period the elimination of the hybrid plasmid does not occur. Moreover, the absence in strain B. anthracis 55ΔTPA-1Spo ^- plasmid area AtxA upregulation allows to obtain the maximum amount of protective antigen without additional material costs and efforts to create an increased concentration of carbon dioxide in the atmosphere. Consequently, the use of B. anthracis strain 55ΔTPA-1Spo ^- to produce the protective antigen and protein EA1 anthrax microbe considerably increases the profitability of the production process of chemical anthrax vaccines.

Concentration and diafiltration of preparations of protective antigen and EA1 protein using membrane filters with an exclusion limit of proteins with a molecular weight of 30 kDa, as well as two-stage chromatographic purification procedures on various carriers provide highly purified antigens that effectively interact with innate and adaptive immunity structures, non-reactogenic and non-reactogenic toxic to the macroorganism.

The possibility of carrying out the invention is confirmed by example 1.

Example 1. Isolation and purification of the protective antigen and protein of the S-layer of the anthrax microbe from the strain B. anthracis 55ΔTPA-1Spo ^-

A pilot fermenter with a working volume of 14 liters is filled with 5 liters of Hottinger broth (pH 7.2-7.4), the medium is sterilized. After sterilization, sterile tryptone solution is additionally added to a final concentration of at least 20 mg / ml.

The preparation of the culture of the producer strain to obtain a sufficient amount of biomass at a certain stage of growth is as follows. One complete loop of the 18-hour agar culture of the producer strain is seeded in test tubes with Khottinger agar containing 25 kg / ml kanamycin and incubated at 37 ° C for 6 hours. After the incubation period, the biomass is washed off by adding 1 ml of chilled 0.85% sodium chloride solution (pH 7.2), resuspended and transferred into bottles with Hottinger beveled agar containing a selective antibiotic in the same concentration. Crops are incubated at a temperature of 37 ° C for 18 hours. The microbial mass is washed off the agar surface by adding 3 ml of chilled 0.85% sodium chloride solution (pH 7.2) to each vial.

For scalable cultivation, 50 ml of cell suspension of the producer strain B. anthracis 55ΔTPA-1Spo ^- with a concentration of microbial cells of at least 50 billion MK / ml, is introduced into the prepared sterile medium. Cultivation is carried out at a temperature of 37 ± 0.1 ° C for 16 hours with constant stirring at 150 rpm After 16 hours, the level of production of protective antigen according to enzyme immunoassay is 640 μg / ml, which is 20 times more than the values determined for the vaccine strain B. anthracis STI-1 (figure 1).

After the growing period, add 2 mm EDTA. Cell mass separation and sterilization is carried out at room temperature and an operating pressure of about 2 bar in a protein concentration and purification unit (Sartorius, Germany) using a Vivaflow 200 tangential filtration module (Sartorius, Germany) containing a membrane filter with a pore diameter of 0.2 μm . After plating 0.1 ml of culture filtrate on plates with Hottinger agar and incubation at 37 ° C for 10 days, the colony morphology characteristic of the anthrax microbe was not observed.

To isolate the protective antigen after sterilization, the culture filtrate is diluted in a 1: 1 volume ratio with a buffer containing 25 mM diethanolamine, 50 mM NaCl, 2 mM EDTA (pH 8.9). It is then concentrated approximately 20 times on a protein concentration and purification unit by filtration in a Vivaflow tangential flow (operating pressure of about 2.5 bar) using a Vivaflow 200 module (Sartorius, Germany) with an exclusion limit of proteins with a molecular weight of 30 kDa. The next step is diafiltration with a 10-fold volume of the same buffer on the same module at room temperature. A concentrated and diafiltered preparation containing a protective antigen is purified on a Macro Prep 50Q chromatographic column (Bio-Rad, USA). The carrier is pre-degassed and washed with the same volume of buffer containing 1M NaCl. The column is then equilibrated by passing successively 10 volumes of a buffer containing 25 mM diethanolamine, 50 mM NaCl, 2 mM EDTA (pH 8.9) and 1 volume of the same buffer with 30 mM KCl (pH 8.9) added. Proteins of the culture filtrate are eluted at a flow rate of 10 ml / min using one free column volume, after which the column is washed with the last buffer in a volume corresponding to the volume of the medium. The resulting sample is combined with the eluate. The protective antigen preparation is concentrated 10 times on the Vivaflow 200 module (30 kDa) at an operating pressure of about 2 bar and room temperature. Diafiltration is carried out on the same module with sequential use of a 10-fold buffer volume containing 25 mM diethanolamine, 50 mM NaCl, 2 mM EDTA (pH 8.9) and a 10-fold buffer volume containing 145 mM ammonium acetate, 2 mM EDTA ( pH 10.0). Before the final purification step, the protective antigen preparation is filtered through membrane filters with a pore diameter of 0.2 μm (Sartorius, Germany), poured into aliquots and stored at minus 70 ° С. Further purification is carried out on a BioLogic Duo Flow ^™ chromatographic system (BioRad, USA). As a carrier for gel filtration, Sephacryl-HR300 (BioRad, USA) is used. The corresponding column is filled with a carrier and equilibrated with 3 volumes of a buffer solution containing 0.1 M TrisOH, 1 mM EDTA, 50 mM NaCl (pH 8.0). A protective antigen preparation cooled to 4 ° C (with a protein concentration of up to 2.5 mg / ml) is applied to the column portionwise in a volume of not more than 3% of the gel volume. The chromatographic system is programmed for a flow rate of 2 ml / min. The yield of proteins is controlled by spectrophotometry at a wavelength of 280 nm. Samples corresponding to the maximum absorption peak are collected and combined after several similar purification cycles. Considering the dilution of the preparation that occurs during gel filtration, the combined samples are concentrated 10 times using Vivaspin-20 concentrators (Sartorius, Germany). The degree of purification of protective antigen, determined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, was 90%. The results of purification of protective antigen are presented in figure 2, where: 1 - culture filtrate of B. anthracis 55ΔTPA-1Spo ^- ; 2 - preparation after concentration on the Vivaflow 200 module (30 kDa); 3 - preparation after cleaning on a column with Macro Prep 50Q; 4 - drug after diafiltration; 5 - preparation after secondary concentration on the Vivaflow 200 module (30 kDa); 6 - molecular weight markers (260, 135, 95, 72, 52, 42, 34, 26, 17, 10 kDa); 7 - the final preparation of protective antigen after chromatography on a column with Sephacryl-HR300. The protective antigen preparation is poured into aliquots and stored at a temperature of minus 70 ° C, and after lyophilization at a temperature of 8 ° C.

To isolate EA1, the cell mass precipitated by centrifugation is washed from the residual medium with a 0.9% sodium chloride solution. 500 ml of biomass is mixed with 50 ml of extraction buffer containing 5 mm Tris-HCl, 1% sodium dodecyl sulfate, 5 mm 2-mercaptoethanol. The mixture is heated in a water bath at a temperature of 70 ° C for 30 minutes, then cooled to room temperature and centrifuged for 30 minutes at 10,000 rpm and a temperature of 4 ° C. The cell extract is sterilized by passing through a Vivaflow 200 tangential filtering module (0.2 μm) at an operating pressure of about 2 bar. In the next step, the cell extract is diafiltered with 10 volumes of buffer containing 0.1 M Tris-HCl, 2 mM EDTA (pH 8.0) at room temperature using a Vivaflow 200 module (30 kDa) under a pressure of about 2 bar. Prior to the purification step, a preparation containing EA1 is stored at a temperature of minus 70 ° C. The purification is carried out on a BioLogic Duo Flow ^™ chromatographic system (Bio-Rad, USA). The column is filled with pre-decanted hydroxyapatite and balanced with 10 free volumes of buffer containing 5 mM K ₂ HPO ₄ · KH ₂ PO ₄ (pH 6.8) at a flow rate of 10 ml / min until a stable baseline is obtained. A preparation containing EA1 was applied to the column and a linear gradient of potassium phosphate (0 to 1 M K ₂ HPO ₄ · KH ₂ PO ₄ ) was created using 3 free column volumes. The fractions corresponding to the absorption peaks at 280 nm are collected and combined. Then, a second purification step is carried out on the same carrier, creating a gradient of potassium phosphate, as described above. The purified EA1 protein is diafiltered with 10 volumes of bidistilled water at 4 ° C using a Vivaflow 200 module (30 kDa) under a pressure of about 2 bar. The degree of purification of EA1 protein, determined by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate, was 95%. The results of purification of EA1 protein are presented in figure 3, where 8 is a cell extract after incubation in a buffer with 1% sodium dodecyl sulfate; 9, 10 - the drug after the first stage of purification on hydroxyapatite; 11 - the final preparation of EA1 protein after the second stage of purification on hydroxyapatite; 12 - molecular weight markers (116.0; 97.0; 66.0; 45.0; 29.0 kDa). If necessary, the EA1 protein preparation is concentrated, poured into aliquots and stored at a temperature of minus 70 ° C, and after lyophilization, at a temperature of 8 ° C.

Thus obtained preparations of protective antigen and protein EA1 are suitable for use as the main and additional components of anthrax chemical vaccine, which is confirmed by the following examples.

Example 2. Determination of the ability of drugs based on protective antigen and EA1 protein to protect laboratory animals when infected with a test strain of the anthrax pathogen

Laboratory animals are immunized with the protective antigen and EA1 protein obtained by the method described above (Example 1). Individual groups of BALB / c mice (20 animals per group) are injected twice with a protective antigen at a dose of 10 μg or a complex preparation containing a protective antigen at the same dose and EA1 protein at a dose of 5 μg. Immunobiological restructuring is assessed by LD ₅₀ indices of the test strain B. anthracis 71/12 and immunity indices. As a result, the LD ₅₀ value of the infecting strain for non-immunized individuals is 5 × 10 ² (100 ÷ 2512) spores; for immunized with protective antigen - 7.9 × 10 ⁴ (15849 ÷ 501187) spores; for those immunized with a complex drug - 1.9 × 10 ⁵ (39811 ÷ 1,000,000) spores. Accordingly, the immunity indices are as follows: 158.5 for the protective antigen, 398.2 for the protective antigen with EA1. Thus, the addition of EA1 to an immunizing preparation based on a protective antigen increases the resistance of linear mice to infection with a test strain of the anthrax pathogen by 2.5 times.

Equal groups of guinea pigs are injected twice with a protective antigen in combination with Freund's complete adjuvant or a protective antigen in combination with EA1 protein and adjuvant. The immunizing dose of protective antigen for guinea pigs is 25 μg / ml, EA1 protein is 10 μg. Immunization with the vaccine strain B. anthracis STI-1 (reference drug) is carried out once subcutaneously at a dose of 5 × 10 ⁷ spores. As a result, the LD _{50 of the} test infection strain B. anthracis 71/12 for control animals is 3.1 × 10 ² spores; for guinea pigs immunized with protective antigen, protective antigen with EA1 and vaccine strain B. anthracis STI-1 1.9 · 10 ⁷ spores. Accordingly, the immunity index in all cases is 100072.0. That is, double immunization of biomodels with a protective antigen or a protective antigen with EA1 provides protection against infection with the test strain B. anthracis 71/12, comparable with the protection of the live anthrax vaccine B. anthracis STI-1. Thus, the proposed method allows to obtain antigenic preparations that effectively protect against infection by the causative agent of anthrax.

Example 3. Determining the ability of purified preparations of protective antigen and EA1 protein to interact with innate immunity structures - toll-like receptors

BALB / c mice were immunized once subcutaneously with a protective antigen (10 μg) or EA1 protein (10 μg) obtained by the method described above (Example 1). Animals are killed after 4 hours, on the 1st, 3rd, 7th and 14th day after immunization and material is taken from the organs of central and peripheral immunogenesis (thymus and spleen). Isolation of RNA from spleen and thymus cells is carried out using the RIBO-sorb reagent kit (InterLabService, Russia) according to the manufacturer's instructions. Reverse transcription is performed using the Revert kit (InterLabService, Russia) in accordance with the manufacturer's instructions. PCR was carried out with the obtained cDNA with specific primers for toll-like receptors (TLRs) of type 2 and 6. A positive control is an amplified fragment of the β-actin “housekeeping” gene. Amplification of gene fragments is carried out on a Tertsik programmable thermocycler (DNA technology, Russia) according to the scheme: one cycle at 95 ° C for 5 min, 30 cycles at 95 ° C for 1 min, 60 ° C for 1 min , temperature 72 ° С - 1 min and the final cycle of 5 min at a temperature of 72 ° С. Electrophoretic analysis of PCR products was carried out in a horizontal chamber (Bio-Rad, USA) in a 2% agarose gel. To determine the size of amplicons, commercial molecular weight markers “O 'GeneRuler 100 bp DNA Ladder Plus ready-to-use (Fermentas, Lithuania) were used. Visualization of DNA bands is carried out using an ultraviolet light source and recorded using a gel documentation system (Bio-Rad, USA). As a result, in all samples taken from 4 hours to 14 days, in immunized animals, in contrast to control animals, a significant increase in the expression of type 2 and 6 TLRs on spleen and thymus cells was noted.

Example 4. Determination of the toxicity of purified preparations of protective antigen and protein EA1 in experiments in vivo and in vitro

Guinea pigs (10 animals per group) are injected subcutaneously once with a protective antigen or EA1 protein in doses of 25, 50, and 100 μg. During the first two weeks with a frequency of once every two days, the animals are weighed and examined. As a result, during 21 days of observation in guinea pigs, visual and palpation changes at the injection site were not observed, the body weight of the animals did not decrease.

To assess the damaging effects in vitro, the protective antigen and EA1 protein at doses of 10 μg are incubated with 3 ml of whole defibrinated blood of healthy donors at 37 ° C for 24 hours. Leukocyte samples are stained with a mixture of mitramycin (125 μg / ml) and ethidium bromide (25 μg / ml). The cytotoxic effect of antigenic drugs is controlled by the number of cells in a state of apoptosis using flow cytofluorimetry. As a result, the purified recombinant anthrax of the anthrax microbe does not increase apoptotic activity and does not have a damaging effect on human blood leukocytes.

To study the proliferative activity of lymphocytes of the organs of central and peripheral immunogenesis, BALB / c mice (6 animals per group) were immunized subcutaneously with a protective antigen or EA1 protein in doses of 10 μg. The controls are intact animals. Thymus and spleen samples are taken after 4 hours, on the 1st, 3rd, 7th and 14th day after immunization. The percentage of thymocytes and splenocytes in the phases of cell division is recorded using a flow cytometer. Apoptosis is assessed by the accumulation of hypodiploid cells in a peak located to the left of the peak corresponding to diploid cells. Activation of immunocompetent cells reflects their number in the proliferation stage. The ratio of cells undergoing apoptosis and proliferation is also determined. The value of this index must be less than one.

As a result, at all stages of the study, the apoptotic and proliferative activity of thymus cells does not significantly differ from similar indicators in the control group. The balance of apoptosis and proliferation does not exceed 1.0, which indicates the absence of the damaging effect of the studied drug on immunocompetent cells. In samples of the spleen taken after 4 hours, on days 1 and 3, the number of proliferating splenocytes corresponded to control values. On days 7 and 14, an increase in the proliferative activity of spleen cells was recorded, which may be due to proliferation of B cells and antibody production. The administration of protective antigen and EA1 protein to mice does not affect the apoptotic activity of splenocytes. The index of the ratio of spleen cells in the stage of apoptosis and proliferation does not exceed 1.0.

Thus, the preparations of the protective antigen and EA1 protein obtained using the above method do not have a damaging effect on the immunocompetent cells of the organs of the central and peripheral immune systems of the macroorganism.

Example 5. Pathomorphological and histological examination of organs of laboratory animals immunized with a protective antigen and protein EA1

Guinea pigs are given a single dose of a protective antigen at a dose of 50 μg in combination with EA1 protein at a dose of 20 μg. Organ harvesting for histological examination is carried out on 1, 3, 7, 21, 28 days after immunization.

Subcutaneous administration of anthrax antigens to guinea pigs does not cause the death of animals and does not affect their general condition. At the injection site after immunization, minimal changes in the form of focal moderate edema of subcutaneous fat and minor lymphocytic infiltration of the dermis, which completely disappear after 3 days, are noted. Changes in the adrenal glands observed during the first 7 days indicate a mild stress reaction. On the part of the parenchymal organs during a macroscopic examination for the entire observation period, no gross dystrophic changes, infiltrative processes, or a sharp violation of the blood supply to the vessels are noted. During the period from 1 to 7 days, histological examination reveals signs of moderate functional tension of the liver parenchyma cells, kidneys, cardiomyocytes against the background of focal phenomena of vascular congestion. Changes in the glomerular apparatus of the kidneys are characterized by moderate plethora of the capillary loops of the vascular glomeruli of the renal corpuscles. The number of renal bodies with changes does not reach 50%. In the liver of guinea pigs in the early stages (1-3 days), moderate congestion is observed in the circulatory system (intralobular sinusoidal hemocapillaries) and the outflow system (central veins) of the blood, combined with the functional tension of the light hepatocytes in the center of the hepatic lobules. However, by day 7, the functional state of the organ does not significantly differ from that observed in animals from the control group.

In lymphoid organs, an increase in activity is recorded from 3 to 21 days, with a maximum reaching on day 27, which indicates the activation of the immune system. From the side of the central organ of the immune system - the thymus, an increase in organ mass by a factor of 2 relative to the mass of the organ in control animals is observed on the 21st day of observation. A 2-fold increase in organ mass is recorded in the spleen with respect to the same indicator in intact animals and moderate hyperplasia of follicular structures (appearance of granularity) on the 21st day of observation. Severe hyperplasia of regional lymph nodes is noted from 21 days. The mass of distant lymph nodes reaches its maximum values on 21 days, during this period it is 30 times higher than the value of the control indicator. However, by the 27th day, the size and mass of the lymph nodes returned to the control values. During histological examination in the thymus with a normal ratio of cortical and medulla, moderate hyperplastic processes from the lymphoid elements and the relative activation of mitotic activity in the medulla begin after 7 days, gradually decreasing by the 27th day. The functional state of the organs of the peripheral immune system fits into the picture of immunogenesis, which was manifested by the sequential activation of T and B zones in them. Thus, the changes revealed by histological examination of the internal organs of guinea pigs immunized with a protective antigen in combination with EA1 protein are permissible and reversible. On the part of immunocompetent organs, manifestations of immunogenesis are recorded.

Thus, the claimed method allows to effectively and without risk of contamination of premises and equipment with anthrax pathogen spores to obtain preparative amounts of highly immunogenic and non-toxic protective antigen and EA1 protein, which are necessary to create specific prophylaxis of anthrax.

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Claims (1)

A method for producing a protective antigen and EA1 protein from B. anthracis strain, comprising preparing a seed culture of a producer strain; cultivation of the asporogenous recombinant strain B. anthracis 55ΔTPA-1Spo ^- at a temperature of 37 ± 0.1 ° C for 16 hours in a liquid nutrient medium containing tryptone with a concentration of at least 20 mg / ml, with constant stirring at 150 rpm; separating the cell mass using a filtration module with a membrane having a pore diameter of 0.2 μm; the isolation of EA1 protein from the washed cell mass and its purification using successive incubation steps with extraction buffer containing 1% sodium dodecyl sulfate, diafiltration using membrane filters with a protein exclusion limit of 30 kDa and two-stage ion exchange chromatography on hydroxyapatite, as well as anti-protective from the culture filtrate and its purification using successive stages of concentration and diafiltration using membrane filters with pre the exclusion of proteins with a molecular weight of 30 kDa and two-stage chromatography on an ion-exchange carrier and a carrier for gel filtration.

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